摘要
以里氏木霉Trichoderma reesei重要工业生产菌Rut C30为出发菌株,通过等离子体(ARTP)诱变筛选,以5‐氟乳清酸(5‐FOA)和尿苷(Uridine)进行筛选,获得一株pyr4基因缺陷株Rut C30ΔU3。用含有野生型里氏木霉pyr4基因的互补质粒转化突变株,可回复野生性状。经测序发现其pyr4基因在核酸序列多个位点发生突变,其中包括两个错义突变和一个移码突变,从而导致乳清酸核苷‐5′‐磷酸脱羧酶失活。经遗传稳定性研究分析,传代5次后仍保持良好的尿苷依赖性、去葡萄糖阻遏以及高产纤维素酶特性。经实验筛选获得了pyr4基因缺陷菌株可作为基因表达系统的受体菌株,建立了以尿苷营养缺陷为筛选标记的木霉转化系统。
An uridine auxotrophic mutant of Trichoderma reesei RutC30 was isolated by resistance to 5-fluoroorotic acid through Atmospheric Room Temperature Plasma mutagenesis. The mutant, called RutC30AU3, recovered to uridine prototrophy by transformation with the wild type T. reesei pyr4 gene cassette. Sequence analysis of this mutated gene ndicated that two missenses mutation and one frame-shift mutation had occurred. Mitotic stability analysis of the mu- tant strain RutC3OAU3 showed a quite good genetic stability. In this study, the pyr4 mutant RutC30AU3 was successfully established and proved to be an excellent recipient strain for gene expression
出处
《菌物学报》
CAS
CSCD
北大核心
2014年第6期1272-1280,共9页
Mycosystema
基金
国家自然科学基金(No.30970073)
国家重点基础研究发展规划(973项目)(No.2011CB707402)
国家高技术研究发展计划(863计划)(No.2012AA092103)
关键词
里氏木霉
等离子体
诱变
pyr4基因
Trichoderma reesei, atmospheric room temperature plasma, mutation, pyr4 gene
